The objective of this proposal is to precisely define viral and cellular determinants of reovirus entry into cells. Mammalian reoviruses are non- enveloped viruses that enter cells by receptor-mediated endocytosis. Reovirus entry requires endosomal acidification and proteolysis of viral outer-capsid proteins. However, the specific mechanisms by which these processes facilitate entry are not understood. An integrated research program using mutant viruses and cells altered in viral entry is proposed to define mechanisms that promote reovirus entry. The research is divided into two parts. In part one (Specific Aims 1 and 2), viral determinants of entry will be defined using mutant viruses selected during persistent infection and mutant viruses selected for resistance to either ammonium chloride or the protease inhibitor E64. Molecular mechanisms of mutant viral phenotypes will be established using reassortment genetics, nucleotide sequence analysis, and site-directed mutagenesis of expressed outer-capsid proteins. Three-dimensional structures of wild-type and mutant viruses will be compared using electron cryomicroscopy and computer image analysis to define structural alterations associated with entry- enhancing mutations. In part two (Specific Aims 3 and 4), cellular determinants of entry will be defined using mutant cells selected during persistent infection and mutant cells selected by gene-trap retroviruses. Blocks to viral entry in mutant cells will be identified by comparing parental and mutant cells for the capacity to support individual entry steps(attachment, internalization, disassembly, and penetration) and for defined aspects of endocytic function (ligand uptake, pH, and proteolytic activity). Host genes required for reovirus entry will be identified by using gene-trap retroviruses as insertional mutagens to isolate cellular libraries in which all expressed, non-essential genes are disrupted by pro-virus integration. These libraries will be selected for resistance to reovirus infection and screened for cells with blocks to reovirus entry. Candidate genes will be identified by plasmid rescue using the pro-virus as a shuttle vector. This research will reveal fundamental mechanisms by which viral and cellular factors cooperate to facilitate reovirus entry and illuminate new target for therapy against viruses that use the endocytic pathway to enter cells.